In this application, we propose to study basic questions about the process of gene amplification in mammalian cells, and how it relates to the physical and functional organization of the genome. We have developed a methotrexate-resistant (MTXr) Chinese hamster ovary cell line (CHOC 400) that has amplified the dihydrofolate reductase (DHFR) gene and approximately 110 kb of flanking DNA about 1,000 times. We have been able to show that initiation of DNA synthesis within the 135 kb amplified DHFR domain (amplicon) occurs at a precise location. We have cloned and mapped approximately 120 kb from the amplicon, including a cosmid containing the initiation locus. Our data indicate that this cosmid contains the origin of DNA synthesis that controls replication of the entire DHFR amplicon. We therefore believe that the unit of amplification is equivalent to a replicon. By comparing the restriction patterns of DNA from independently-derived MTXr Chinese hamster cells, we have obtained evidence that the amplicon in all these cell lines may be the same size and have similar restriction patterns. We therefore want to test the hypothesis that the origin site of amplification (i.e., the boundaries of the amplicon) is fixed by sequences in the parental DNA which normally serve some function in the cell (e.g., initiation or termination of DNA synthesis). We propose: 1) to screen for and characterize additional recombinant cosmids in order to isolate the entire amplicon from CHOC 400; 2) to utilize these cosmids as radioactive probes on genomic digests of various MTXr Chinese hamster cell lines to establish whether the amplicon is identical among independently-derived Chinese hamster cell lines; 3) to prepare a cosmid library from the parental CHO cell in order to isolate fragments representing the original site of amplification, and to determine the location of the junctions relative to the origin of DNA synthesis; 4) to determine whether the origin used in the CHOC 400 amplicon is the same one utilized in the parental cell to replicate the DHFR domain; 5) to transform Chinese hamster cells with the cloned DHFR gene, and to establish cell lines that have amplified this gene in a new chromosomal location; the amplicon in these cell lines will then be analyzed for size and restriction pattern, and for the presence of an origin of DNA synthesis or homologies to other amplicons.